Detection of potential cross-reaction between a short oligonucleotide sequence and a longer (unintended) sequence is crucial for many biological applications, such as high content screening (HCS), microarray nucleotide probes, or short interfering RNAs (siRNAs). However, owing to a tolerance for mismatches and gaps in base-pairing with target transcripts, siRNAs could have up to hundreds of potential target sequences in a genome, and some small RNAs in mammalian systems have been shown to affect the levels of many messenger RNAs (off-targets) besides their intended target transcripts (on-targets). The reference sequence (RefSeq) collection aims to provide a comprehensive, integrated, nonredundant, well-annotated set of sequences, including mRNA transcripts. We performed a detailed off-target analysis of three most commonly used kinome siRNA libraries based on the latest RefSeq version. To simplify the access to off-target transcripts, we created a SeedSeq database, a new unique format to store off-target information.